The studies proposed in this application are designed to examine the mechanism and regulation of actinomcyin synthesis in Streptomyces antibioticus. In previous experiments, the structural gene for phenoxazinone synthase (PHS), a key enzyme in the actinomycin biosynthetic pathway was cloned. In the present application it is proposed to: (1) Characterize a recently isolated DNA fragment which overlaps the ends of the PHS gene for sequences reponsible for glucose catabolite repression of PHS synthesis. Techniques of in vitro mutagenesis will be used to identify those sequences: (2) Clone the gene for the enzyme which activates 4-methyl-3-hydroxyanthranilic acid, the precursor of the phenoxazinone chromphore in the actinomycin biosynthetic pathway: (3) Characterize the clones obtained in (2) using molecular biological and biochemical techniques; (4) Examine the possible regulation of gene expression in S. antibioticus by RNA polymerase. For these studies, experiments will be designed to examine the possible specificity of RNA polymerase sigma factors in the transcription of S. antibioticus genes. The proposed studies are significant because they will provide information relating to the regulation of antibiotic synthesis in particular and secondary metabolism in general in an extremely interesting class of organisms. The proposed research will also provide information on gene organization and expression generally in prokaryotes. Finally, the proposed studies may lead to a level of understanding of the mechanisms of antibiotic production which will allow the use of genetic engineering techniques to construct organisms which are capable of hyperproduction of existing antibiotics or the production of new antibiotics. Thus, the proposed studies will have potential applications to industry and to clinical and veterniary medicine.